Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States.
ACS Chem Biol. 2022 Sep 16;17(9):2583-2594. doi: 10.1021/acschembio.2c00511. Epub 2022 Aug 29.
Apurinic/apyrimidinic endonuclease-1 (APE1) is a base excision repair (BER) enzyme that is also engaged in transcriptional regulation. Previous work demonstrated that the enzymatic stalling of APE1 on a promoter G-quadruplex (G4) recruits transcription factors during oxidative stress for gene regulation. Also, during oxidative stress, cysteine (Cys) oxidation is a post-translational modification (PTM) that can change a protein's function. The current study provides a quantitative survey of cysteine oxidation to sulfenic acid in APE1 and how this PTM at specific cysteine residues affects the function of APE1 toward the 3 gene promoter G4 bearing an abasic site. Of the seven cysteine residues in APE1, five (C65, C93, C208, C296, and C310) were prone to carbonate radical anion oxidation to yield sulfenic acids that were identified and quantified by mass spectrometry. Accordingly, five Cys-to-serine (Ser) mutants of APE1 were prepared and found to have attenuated levels of endonuclease activity, depending on the position, while values generally decreased for G4 binding, indicating greater affinity. These data support the concept that cysteine oxidation to sulfenic acid can result in modified APE1 that enhances G4 binding at the expense of endonuclease activity during oxidative stress. Cysteine oxidation to sulfenic acid residues should be considered as one of the factors that may trigger a switch from base excision repair activity to transcriptional modulation by APE1.
脱嘌呤/脱嘧啶核酸内切酶-1（APE1）是一种碱基切除修复（BER）酶，也参与转录调控。先前的工作表明，APE1 在启动子 G-四链体（G4）上的酶促停滞在氧化应激期间招募转录因子进行基因调控。此外，在氧化应激过程中，半胱氨酸（Cys）氧化是一种可以改变蛋白质功能的翻译后修饰（PTM）。本研究定量调查了 APE1 中半胱氨酸氧化为亚磺酸的情况，以及这种特定半胱氨酸残基上的 PTM 如何影响 APE1 对带有碱基缺失的 3 个基因启动子 G4 的功能。在 APE1 的七个半胱氨酸残基中，有五个（C65、C93、C208、C296 和 C310）容易被碳酸盐自由基阴离子氧化生成亚磺酸，通过质谱鉴定和定量。因此，制备了 APE1 的五个 Cys-to-serine（Ser）突变体，发现其内切酶活性根据位置减弱，而 G4 结合的 值通常降低，表明亲和力增加。这些数据支持这样的概念，即半胱氨酸氧化为亚磺酸会导致修饰的 APE1，在氧化应激期间以牺牲内切酶活性为代价增强 G4 结合。半胱氨酸氧化为亚磺酸残基应被视为可能触发 APE1 从碱基切除修复活性向转录调节转换的因素之一。
